The mammalian phosphoglycerate kinase (PGK) gene family remains unparalleled in the number of different facets of gene regulation that impinge upon the expression of these genes in spermatogenesis. The two functional PGK genes are differentially expressed in spermatogenesis. The X-linked PGK-1 gene is ubiquitously expressed at low levels in the premeiotic germ cells, as well as in all somatic cells, while the autosomal PGK-2 gene is specifically expressed at high levels in the postmeiotic stages of spermatogenesis, were the PGK-1 gene is inactivated. Thus in this system the trnscriptional activation of a tissue-specific gene (PGK-2) is coincident with the inactivation of an analogous housekeeping gene (PGK-1), both of which produce a similar protein product (phosphoglycerate kinase). It is the goal of this project to discern the regulatory mechanisms governing the differential expression of the PGK genes in spermatogenesis. This will be done first by more precisely delineating the expression patterns of each PGK gene at the RNA level in spermatogenesis, using in situ cytohybridization on tissue sections and/or Northern hybridization to RNA from sorted spermatogenic cells. Subsequently, and analysis of the 5'-promoter sequences of each PGK gene will be done to identify any specific sequences involved in cell-type-specific expression. Also a search for cell-type-specific proteins that bind to these promoters will be used to identify key transcription factors that regulate expression. An analysis of the role of methylation in gene regulation will also be carried out on each of the PGK genes isolated from expressing versus non-expressing spermatogenic cells. Finally, the intronless structure of the spermatogenic-specific PGK-2 gene will be compared to that of the ubiquitously expressed intron-containing PGK-2 gene, with special attention to the possible effect the presence or absence of intervening sequences may have on gene expression in this system. Two extensions of this recently reviewed research project are also proposed. One takes advantages of our recently developed ability to isolate large, pure populations of primordial germ cells (PGC) from early mouse embryos. Thus an analysis of PGK gene structure and expression can now be performed on this early stage in germ cell development. The second involves analysis of PGK gene structure and expression in spermatogenic cells of the chicken. Because there is only a single PGK gene in this species, a comparison of this system with the two-gene system in mammals should shed light on both the function and evolution of the mammalian PGK genes.